Seligman J, Newton GL, Fahey RC, Shalgi R, Kosower NS. Nonprotein thiols and disulfides in rat epididymal spermatozoa and epididymal fluid: role of γ-glutamyl-transpeptidase in sperm maturation. J Androl. 2005;26:629–637.
In the age of KOs, siRNAs, and arrays, studies are often driven by the temptation of trying new techniques, and, as a result, basic physiological questions can become overlooked. However, there is no substitute for studies aimed at understanding the physiologic processes in an intact organism. This is exactly the approach taken by Seligman et al (2005) in their elegant study reported in this issue of the Journal of Andrology, where they propose a novel mechanism for stabilization of sperm thiols in the epididymis.
In their study, Seligman et al use the γ-glutamyl transpeptidase (γ-GT) inhibitor acivicin to show that the enzyme γ-GT is actively involved in catabolizing glutathione (GSH) to cysteine (CSH) in the caput epididymidis. Furthermore, they propose that the oxidative potential generated by this reaction facilitates oxidation of free thiols, thus forming GSSG, GSSC, and CSSC moieties.
The Figure summarizes the redox reactions that occur as a result of γ-GT activity in the epididymal lumen (Drozdz et al, 1998; Paolicchi et al, 2002). These reactions, along with the activity of sperm NADPH oxidase, generate a number of different reactive oxygen species (ROS). Therefore, as is suggested in Seligman et al (2005), there may be sufficient ROS in the epididymal lumen to drive autoxiation of GSH and CSH.
However, apart from thiol auto-oxidation, GSSG might also be generated through GSH oxidation by the enzyme glutathione peroxidase. This enzyme is known to be secreted and active in the epididymis (Vernet et al, 2004), and it would be reasonable to assume that it is also involved with disulfide formation as spermatozoa traverse the caput epididymidis (Figure). There may be still other enzymes involved in free thiol oxidation in the epididymis. One such possibility mentioned in Seligman et al (2005) is sulfhydryl oxidase, which catalyzes the superoxide-dependent oxidation of GSH and CSH and has also been shown to have high activity in male reproductive tissues (Chang and Zirkin, 1978).
In recent years, γ-GT has emerged as an interesting enzyme to male reproduction. Not only is it extremely abundant in epididymal tissues (Agrawal and Vanha-Perttula, 1988), but also, the null mutation model has shown its presence to be crucial for male fertility (Kumar et al, 2000). Reports have looked at γ-GT promotor regulation in vivo (Kirby et al, 2004) with a focus on epididymis-specific pathways for controlling enzyme expression.
However, despite the interest in γ-GT expression regulation, a key question remained unanswered: what, in fact, is the enzyme's function in the epididymis? Seligman et al (2005) shed light on this issue by demonstrating its active involvement in converting GSH to CSH, particularly in the caput epididymidis. While this is a step toward understanding the role of γ-GT in male reproductive tissues, the findings presented in this article also open the door to further questions about the enzyme's function.
It is known that several γ-GT isoforms exist in the epididymis (Palladino and Hinton, 1994) and that they are subject to segment-specific differential regulation by testicular factors and testosterone (Palladino and Hinton, 1994). It would be of interest to identify which of these isoforms are involved with the activities described by Seligman et al (2005).
Another question that emerges from data presented in the article is the exact location of γ-GT in the epididymis. While the authors state that the enzyme is localized in the epididymal epithelium, the continued conversion of free thiols to disulfides in the presence of epididymal fluid alone suggests that a secreted form of γ-GT also exists. Immunohistologic analysis of this enzyme would be a useful follow-up to what we now know about its role in the epididymis.
One of the particularly novel findings by Seligman et al (2005) is that the presence of oxidized thiols is essential and, perhaps, sufficient for disulfide bond formation in sperm. This poses a question regarding the role of the enzyme hydroperoxide glutathione peroxidase (PHGPx, also known as glutathione peroxidase 4) in sperm maturation. Numerous publications pertaining to the role of PHGPx in sperm nuclear disulfide formation have established the apparent importance of this enzyme to the process (Tramer et al, 2002; Vernet et al, 2004). The study by Seligman et al (2005) opens the possibility of the existence of an alternate pathway to thiol oxidation in sperm. Immunohistologic examination would once again be an interesting approach to help resolve this issue, as staining for both PHGPx and γ-GT could help dissociate the involvement of each enzyme in sperm maturation.
Apart from understanding the function of γ-GT in the epididymis, the study also touches on the question of protein and nonprotein thiols in the sperm nucleus. As illustrated in the Figure, mature sperm have a mixed disulfide population, where protamines can form disulfide bonds either with other protamines or with nonprotein thiols such as GSH and CSH. However, we currently have little knowledge regarding what types of disulfides are present, the reactions by which they are formed, and the relative contributions of each. Future studies on this topic could provide valuable information for building an accurate model of sperm chromatin packaging.
Overall, the findings presented by Seligman et al (2005) are an important step toward understanding sperm nuclear stabilization. They offer a new interpretation of clinical data that correlate infertility and abnormal sperm with both under- or overoxidation of nuclear thiols (Rufas et al, 1991; Evenson et al, 2000; Zini et al, 2001) and suggest the involvement of γ-GT in these pathologies. Furthermore, the study once again reminds us of the duality of oxidative processes; antioxidants can turn into oxidants, which drive sperm thiols to form stable relationships.