Seminal plasma antioxidant activity

In order to prevent possible cellular damage, excess ROS are constantly scavenged to maintain low concentrations essential for vital cell functions. Seminal plasma is well provided with numerous enzymatic antioxidants including CAT and SOD (Baumber et al., 2000; Fujii et al., 2003; Hsieh et al., 2002). The activity of SOD causes the dismutation of O2−• radicals and their conversion into O2

and H2O2. The H2O2 produced is subsequently detoxified by CAT to produce H2O and O2 (Zelen et al., 2010; Zini et al., 1993).

In this study, seminal plasma CAT activity was correlated significantly and positively with the proportions of total, progressive and rapid motility as well as normal morphology spermatozoa, and significantly and negatively with the proportion of immotile spermatozoa. Similar, but non-significant trends were observed for the SOD activity. These results are in agreement with those reported by Khosrowbeygi et al. (2004), who demonstrated a significant positive correlation between CAT activity and the percentages of progressive motility and normal morphology, while the correlations with SOD activity were not significant. Previous studies have further shown a substantially higher seminal plasma activity of CAT in normozoospermic men as compared to men with asthenozoospermia (Atig et al., 2012; Bykova et al., 2007; Siciliano et al., 2001; Tavilani et al., 2008) or asthenoteratozoospermia (Khosrowbeygi et al., 2004). The observed positive correlations between CAT activity and sperm motility and morphology indicate the importance of this enzyme in the alleviation of ROS-induced oxidative damage, thus reducing the cytotoxicity to spermatozoa.

Available literature provides inconsistent results about the relationship between SOD activity and sperm quality. Some studies have revealed that increased SOD activity in seminal plasma is correlated with a significant improvement in the sperm overall motility (Atig et al., 2012; Murawski et al., 2007; Yan et al., 2014). Other studies have also reported similar, but non-significant results (Hsieh et al., 2002; Khosrowbeygi et al., 2004; Macanovic et al., 2015). The current study did not find a correlation between SOD activity in seminal plasma and sperm motility parameters, while the correlations with VCL and ALH were significantly positive. This suggests that elevated SOD activity in seminal plasma might be an indication of the development of spontaneous premature hyperactivated motility of spermatozoa in the ejaculate. However, sperm regulation is a highly complex process involving multiple variables, thus, the specific role of SOD in the control of sperm motility remains poorly understood and necessitates further research.

The results of the current study exemplify the importance of CAT activity in seminal plasma as a major marker of the antioxidant status of the ejaculate. The ability of CAT to improve sperm motility indicates that H2O2 is the ROS that represents the dominant cytotoxic effect upon spermatozoa. In

contrast, the lack of significant correlations between O2−• levels and sperm motility as well as between SOD activity and sperm motility suggests that O2−• plays a considerably less cytotoxic role in spermatozoa. These observations are consistent with those obtained by Aitken et al. (1993), Armstrong et al. (1999) and Baumber et al. (2000) who concluded that H2O2 is especially responsible for the motility impairment of spermatozoa. These experiments also demonstrated that addition of CAT, but not SOD, to the incubation medium could ameliorate the decline in sperm motility induced by oxidative stress.

There has been a wide consistency among studies about the deleterious effects of H2O2 on the quality of sperm movement, with progressive motility being the most affected parameter (Calamera et al., 2001; Du Plessis et al., 2010; Maia et al., 2014). Moreover, the observed decline in sperm motility associated with increased H2O2 concentrations was reported to occur in the absence of any measurable signs of plasma membrane damage (Baumber et al., 2000; Calamera et al., 2001). This may substantiate the lack of significant correlation between CAT activity in seminal plasma and sperm viability observed in the current study.

Due to its high permeability across biological membranes, H2O2 is assumed to have direct effects on the intracellular enzyme systems (Maia et al., 2014). Several possibilities have been suggested to explain the mechanism through which H2O2 deteriorates sperm motility. However, the exact mechanism appears to be not entirely elucidated and merits further investigations. Some studies have ascribed this effect to the potential capacity of the H2O2 to deplete intracellular ATP levels (Lamirande and Gagnon, 1993; Armstrong et al., 1999; Bilodeau et al., 2002), leading to impaired phosphorylation of axonemal proteins essential for sperm motility. In contrast, Calamera et al. (2001) demonstrated that the loss of sperm motility after incubation with H2O2 was associated with a corresponding increase in intracellular ATP levels due to decreased ATP utilization by non-progressive and immotile spermatozoa. Moreover, excess intracellular concentrations of H2O2 have also been assumed to deteriorate sperm motility throughout the inhibition of intracellular ROS scavenging activity (Krzyzosiak et al., 2000; Maia et al., 2014).

5.2.4 Acrosome Reaction

In order to attain full fertilization potential, spermatozoa undergo an exocytotic process characterized by the release of the acrosome's lytic enzymes to enable the fusion of the spermatozoa outer plasma membrane with the oocyte (Esteves and Verza, 2011). The physiological acrosome reaction is an irreversible event that must occur at an appropriate time during the early stages of sperm-egg interaction. Thereby, premature spontaneous acrosome reaction renders the spermatozoon unable to bind and penetrate an oocyte normally (Liu and Baker, 1994; Tesarik, 1989).

The proportion of acrosome-intact spermatozoa, in the current study, was significantly and positively correlated with the proportions of type C, medium and slow spermatozoa, while its correlations with other semen analysis parameters were statistically not significant. These findings appear to contradict those reported by Parinaud (1996), who found that the proportion of spontaneously acrosome-reacted spermatozoa was negatively correlated with progressive motility. Such variation could possibly be due to the utilization of different techniques for the assessment of acrosome integrity, which allows for the detection of different stages of the acrosome reaction (Köhn et al., 1997). In the above cited study (Parinaud, 1996), the acrosome reaction was evaluated using FTTC-GB24 which primarily binds to the inner acrosomal membrane. This assay detects only spermatozoa that have undergone a complete acrosome reaction (Parinaud et al., 1993), since labelling with GB24 lectin necessitates the exposure of this membrane subsequent to a complete loss of the acrosomal content (Fenichel et al., 1989). However, in the present study, identification of acrosome-reacted spermatozoa was performed using FITC-PSA. The PSA lectin is known to bind with the acrosomal matrix (Risopatron et al., 2001) and thereby allows for the identification of both partially and completely acrosome-reacted spermatozoa (Jaiswal et al., 1999; Ozaki et al., 2002).

The observed positive correlation between sperm acrosome integrity and non-progressive motility may presumingly be attributed to an increased fluid resistance to motion and inertia caused by the sperm with larger acrosomal area. In consonance, previous experiment performed in our laboratory showed a negative correlation between the acrosome size and progressive motility in post-swim-up

samples (Murray, 2007). Although prematurely acrosome-reacted spermatozoa might have the ability for forward motility, they are not able to penetrate the zona pellucida and thereby unable to fertilize the egg.

5.2.5 DNA Fragmentation

Despite the remarkable developments in automated semen analyses, the parameters of the conventional semen analysis remain of relatively limited value in clinical practice (Evgeni et al., 2014; Oleszczuk et al., 2013; Vogiatzi et al., 2013). More valuable information about the quality of sperm as well as pregnancy outcome can be obtained via combining results from the conventional semen analysis and validated sperm DNA fragmentation assays (Borini et al., 2006; Evgeni et al., 2014; Fernández-Gonzalez et al., 2008; Sheikh et al., 2008). In this study, a significant negative correlation was observed between the proportion of DNA fragmentation and sperm viability. This result is comparable with the previous findings from Brahem et al. (2012) that revealed a strong negative correlation exists between sperm DNA fragmentation and the percentage of viable spermatozoa. Higher levels of necrozoospermia were also observed among men with elevated levels of sperm DNA fragmentation. Furthermore, the increase in sperm DNA fragmentation induced by long-term in vitro incubation was reported to be accompanied by a substantial loss of sperm viability (Muratori et al., 2003). Similarly, a more recent study also demonstrated a strong negative correlation between sperm DNA fragmentation and viability in semen samples with DNA fragmentation rates ≥ 30 % (Samplaski et al., 2015). The current study confirms the observations of the above mentioned studies and suggests that sperm viability might represent a potential indicator and a cost-saving measure for semen quality.

Both DNA integrity and viability of spermatozoa are known to be important markers of semen quality. The mechanism responsible for the incidence of DNA fragmentation in ejaculated human spermatozoa is not fully elucidated. One hypothesis proposes DNA breaks within ejaculated spermatozoa to be the result of apoptotic DNA cleavage during the early stages of spermatogenesis (Sakkas et al., 1999). However, at the stage of DNA break down, the apoptotic process is irreversible

and the cells would be eliminated by Sertoli cells prior to ejaculation (Agarwal et al., 2012). Another postulation points to the excessive exposure to ROS as being the causative agent for DNA fragmentation in ejaculated spermatozoa (De Lamirande and Gagnon, 1999). Sperm DNA fragmentation has previously been shown to correlate significantly and positively with the levels of ROS generated by spermatozoa (Barroso et al., 2000). Despite not being able to measure ROS and DNA fragmentation in the same samples, the current study showed a significant negative correlation (r = -0.33; P = 0.04) between sperm intracellular O2−• levels and the proportion of viable spermatozoa, thereby, indirectly implying a relationship between ROS and DNA fragmentation.

The current study also observed a positive correlation between the sperm DNA fragmentation and the kinematic parameters, VCL, LIN and STR. This shows that DNA fragmented spermatozoa might still have the capacity for rapid forward motility. However, these spermatozoa might not be able to develop a state of hyperactivated motility at the site of fertilization as was indicated by the negative correlation observed in this study between the proportion of DNA fragmentation and ALH.

Several studies have been undertaken to investigate the possible correlation between sperm DNA fragmentation and a number of semen characteristics such as sperm concentration, motility and morphology. Not all studies, however, have come to the same conclusions. Some studies have revealed poor correlations, as was observed in the present study, between the amount of sperm DNA fragmentation and the basic semen parameters of sperm concentration, motility and morphology (Cassuto et al., 2012; Chenlo et al., 2014; Giwercman et al., 2003; Karydis et al., 2005; Xia et al., 2005). In contrast, other studies have shown significant negative correlations between sperm DNA fragmentation and many of these semen variables (Lin et al., 2008; Sheikh et al., 2008; Zini et al., 2001). More recently, Boushaba and Belaaloui (2015) reported negative correlations between sperm DNA fragmentation and sperm concentration as well as motility, while no significant correlation was found with regards to sperm morphology. As stated in a review by Evgeni et al. (2014), the inconsistencies among different studies concerning the correlation between sperm DNA fragmentation and semen characteristics could be ascribed to several factors. These factors include

various assays used to quantify DNA fragmentation, the use of different techniques for the assessment of semen quality as well as dissimilarities in the characteristics of the populations across studies. The use of flow cytometry-based TUNEL assay in the current study allows for the simultaneous measurement of real DNA damage in a large population of spermatozoa, providing more objective and statistically reliable outcomes (Chenlo et al., 2014; Sharma et al., 2013). Nevertheless, none of the sperm quality parameters (concentration, motility and morphology) displayed a significant correlation with the proportion of DNA fragmented spermatozoa. The result of this study highlights the importance of the TUNEL assay in predicting sperm function, independent of any quantifiable changes in sperm concentration, motility or morphology as measured by CASA in a population of normozoospermic men.