*3.2. Chemical Factors*

Aside from the physical factors discussed above, male fertility is also sensitive to a variety of toxicants, such as those arising from industrial processes or from common everyday materials, which accumulate in the human body. Indeed, it is well established that the accumulation of the heavy metals lead and cadmium can impair multiple semen parameters, including sperm motility [61]. Similarly, male rats treated with lithium display OS in their testes and experience reduced sperm count and

motility [62]. The induction of OS in the male reproductive tract has also been cited as a causal agen<sup>t</sup> responsible for elevated levels of apoptosis among developing germ cells, defects in sperm morphology and impaired sperm function in mice treated with industrial contaminants used in the production of plastics, such as bisphenol-A [63], mono-butyl phthalate [64] and other related compounds [65]. This spectrum of deleterious OS-related e ffects extends to other forms of chemical exposure including those associated with excessive alcohol consumption or cigarette smoking. In this context, chemicals contained within cigarette smoke have been shown to cause local inflammation, an attendant 48% increase in seminal leukocytes, and a 107% increase in seminal ROS levels [66]. It follows that the semen of cigarette smokers is not only characterized by increased ROS levels, but also extensive molecular changes in the spermatozoa and reduced overall semen quality [67]. Likewise, excessive alcohol consumption can lead to ethanol-induced cell membrane destruction, increased production of ROS and impaired sperm function [68,69].

From the preceding discussion, it is apparent that acute and/or chronic exposure to a variety of external or internal factors can trigger the overproduction of ROS and reduce antioxidant defenses within the male reproductive tract, thus propagating an OS cascade and resulting in LPO (summarized in Figure 1).

**Figure 1.** (**a**) Sources of reactive oxygen species (ROS) in human spermatozoa and the relationship between the rate of their production and antioxidant defenses during oxidative stress. (**b**) ROS are capable of attacking polyunsaturated fatty acids (PUFAs) within cellular membranes, initiating lipid peroxidation cascades (LPO) and resulting in the production of cytotoxic lipid aldehydes such as 4-hydroxynonenal (4HNE). Abbreviations: EME, electromagnetic energy; ETC, electron transport chain.

### **4. ROS-Induced Lesions Detected in Low-Motility Spermatozoa**

With the diverse range of factors that can amplify the levels of ROS in semen, attention has naturally focused on the impact of these powerful oxidants on sperm function. Through decades of research we have come to realize that the highly specialized sperm cell is exceptionally vulnerable to disturbance in ROS levels owing to the presence of modest antioxidant defenses and conversely, a myriad of oxidizable substrates [70]. Not the least of these are the polyunsaturated fatty acids (PUFAs; such as linolenic, arachidonic and decohexaenoic acids) that dominate the lipid architecture of the sperm plasma membrane. In the case of human spermatozoa, the predominant PUFA is decohexaenoic acid (DHA), a lipid that accounts for >50% of all membrane PUFAs [70,71]. PUFAs such as DHA not only play a major role in the regulation of sperm membrane fluidity, but owing to the presence of multiple carbon-carbon double bonds, they also serve as prime substrates for ROS attack. The resultant cascade of lipid peroxidation (LPO) reactions catalyze the formation of numerous breakdown products including a suite of highly reactive lipid aldehydes [e.g., malondialdehyde (MDA), 4-hydroxynonenal (4HNE) and acrolein] [11] (see Figure 1b).

Under physiological conditions, aldehyde-metabolizing enzymes function to detoxify and prevent the accumulation of these advanced end products of LPO [72]. However, excessive OS can promote the accrual of lipid aldehydes and, owing to their inherent stability (relative to that of free radicals), these electrophiles can elicit widespread cellular damage and pathological dysfunction in human spermatozoa [73,74]. In the case of 4HNE, which ranks among the most abundant and cytotoxic of the lipid aldehydes, the chemical structure contains three reactive functional groups: a C2=C3 double bond, a C1=O carbonyl group and a hydroxyl group on C4 [75]. These structural elements render the 4HNE electrophile highly reactive toward nucleophilic groups, enabling the formation of both the Michael addition of thiol or amino compounds and Schiff bases with primary amines. Thus, 4HNE has the ability to react with proteins (principally those containing histidine, cysteine and lysine residues), lipids, and nucleic acids (mostly with the guanosine moiety of DNA) [76–78]. In spermatozoa, the creation of 4HNE adducts has been linked with compromised membrane integrity, motility defects and reduced ability to participate in oocyte interactions [22,73,79]; thus reducing overall fertility. Moreover, studies by Bromfield and colleagues [73,79] have shown that the impact of 4HNE can vary depending on the timing and of the insult. Thus, 4HNE can drive post-meiotic round spermatids towards a ferroptotic cell death pathway, whereas an equivalent exposure of mature spermatozoa can elicit functional lesions, which compromise their fertilization potential (e.g., dysregulation of the molecular chaperone Heat Shock Protein A2 and an accompanying disruption of oocyte recognition), but does not overtly impact their viability [73,79]. Such differential pathogenesis may be attributed to the highly specialized architecture of the male germ cell, which depending on their stage of differentiation, features an abundance of substrates for 4HNE attack, minimal antioxidant defense enzymes, and limited capacity for self-repair when 4HNE-mediated damage is sustained [11]. However, excess ROS production can also directly impact sperm function via increases in redox driven protein modifications [80]. Thus, OS has been shown to promote an increase in S-glutathionylation and tyrosine nitration of sperm proteins, both of which adversely impact motility [80,81]. Similar alterations have been documented by Vignini et al. [82], who demonstrated an increase in ONOO− concentrations and tyrosine nitration in human asthenozoospermic sperm samples.
