Abnormal sperm quality is often related to elevated oxidative stress [1]. For example, high levels of reactive oxygen species (ROS) were measured in 30–80% of men with unexplained infertility [2]. Most of the environmental or lifestyle factors that impair male fertility are also known to induce oxidative stress. If the antioxidant system is insufficient or overwhelmed, testicular function and spermatogenesis become disturbed [3,4]. In line with the detrimental oxidation of biomolecules such as lipids or DNA, oxidative stress also promotes glycoxidation reactions, which result in the formation of highly reactive compounds, such as methylglyoxal (MG) [5], which is also known as a by-product of the metabolic pathways, e.g., glycolysis [6]. An abnormal accumulation of MG and other dicarbonyl metabolites leads to their reaction with proteins, nucleotides, and basic phospholipids, forming advanced glycation end products (AGEs) and results in dicarbonyl stress [7,8]. The reaction of dicarbonyl metabolites with proteins is often directed toward arginine residues, forming dihydroxyimidazolidine and hydroimidazolone adducts and leading, in many cases, to malformed and inactive proteins [6]. Increased levels of nucleotide AGEs are associated with DNA strand breaks and mutagenesis [9]. These AGEs induce adverse effects in the body via non-receptor- and receptor-mediated mechanisms and can contribute to the pathogenesis of some diseases [10,11], e.g., diabetes [7]. The interaction of AGEs with their receptors can also result in ROS generation, as well as the activation of NF-κB and the subsequent transcription of numerous proinflammatory genes [11].

Testis tissue has a high metabolic rate and energy demand because of mitotic and meiotic cell replication. A finely tuned system of energy provision via oxidative phosphorylation and glycolytic activity has evolved to meet the requirements of the various cell types [12]. While ROS may be largely generated as a result of redox reactions within the mitochondria and the action of oxidases in differentiating spermatogenic and somatic cells, glucose is the substrate used to maintain the high glycolytic level required for the self-renewal of spermatogonial stem cells and the survival of spermatozoa [12,13]. Therefore, antioxidant activity is important for the homeostasis of testicular cells [14], but also the enzymes that may detoxify AGEs, which have recently been discussed as a cause of male infertility [15]. The higher glucose level observed in diabetic humans and animals could lead to the higher production of AGEs and subsequent sperm damage [15]. It has been shown that AGEs impair the DNA integrity of human spermatozoa [16]. If cultured rat Leydig cells were treated with AGEs, testosterone production was suppressed, and ROS content increased, signifying the close interconnection between dicarbonyl and oxidative stress [17].

A central compound involved in eliminating ROS, as well as AGEs, is reduced glutathione (GSH). It provides electrons for reducing, e.g., H2O2 to water [18], and it is also reaction partner within the glyoxalase system/MG pathway. In this pathway, highly reactive α-oxoaldehydes, such as MG, react with GSH to produce a thioacetal. In the case of MG as a substrate, glyoxalase I (GLO I) converts the thioacetal to S-D-lactoyl-glutathione (SLG). Catalyzed by the activity of glyoxalase II (GLO II, HAGH – hydroxyacylglutathione hydrolase), SLG may be transformed into d-lactate upon the release of GSH [19]. The homodimer GLO I and the monomer GLO II are the enzymes responsible for avoiding dicarbonyl stress by balancing MG levels. Both are metallo-enzymes with Zn2+ as the frequently detected active site metal ion [19]. In addition to its function in the glyoxalase system, Ercolani et al. suggest an additional function on the part of GLO II, specifically that the enzyme could mediate the S-glutathionylation of specific proteins [20]. This mechanism is, in general, a method of protecting protein thiol groups from irreversible oxidation, regulating protein function, or storing GSH during oxidative stress [21].

Like humans, many felid species also suffer from teratozoospermia and produce only a small number of normal spermatozoa [22,23]. However, nothing is known about the antioxidant or glyoxalase systems in the feline testis and their capacity to mitigate oxidative stress or an excess of AGEs, which represent potential threats. In the present study, we have analyzed the expression of the two glyoxalases in the testes of the domestic cat (Felis catus) on an RNA level in the whole testis tissue, as well as on the protein level via immunohistochemistry. We investigated testes in different developmental stages, which differ in their characteristic cell composition and spermatogenic activity. In prepubertal samples, spermatogonia are the only germ cells. During puberty, differentiation and meiosis are initiated, with round spermatids being the most advanced spermatogenic cell stage. Spermatogenesis is completed in postpuberty I and most intensified in postpuberty II samples [24].