Regucalcin counteracts tert-butyl hydroperoxide and cadmium-induced oxidative stress in rat testis Sara Correia, Cátia V. Vaz, Ana M. S. Silva, José E. Cavaco and Sílvia Socorro* ABSTRACT: Regucalcin (RGN) is a calcium (Ca 2+ )-binding protein with multiple physiological roles and has also been linked to the suppression of oxidative stress. It is widely known that oxidative stress adversely affects spermatogenesis, disrupting the devel- opment of germ cells, and interfering with sperm function. The present study aims to analyze the role of RGN modulating testic- ular oxidative stress. To address this issue, seminiferous tubules (SeT) from transgenic rats overexpressing RGN (Tg-RGN) and wild-type (WT) were cultured ex vivo for 24 h in the presence/absence of pro-oxidant stimuli, tert-butyl hydroperoxide (TBHP, 250 and 500 μM) and cadmium chloride (Cd, 10 and 20 μM). Noteworthy, SeT from Tg-RGN animals displayed a significantly higher antioxidant capacity and diminished levels of thiobarbituric acid reactive substances relatively to their WT counterparts, both in control and experimental conditions. Regarding the antioxidant defense systems, a significant increase in the activity of glutathione-S-transferase was found in the SeT of Tg-RGN whereas no differences were observed in superoxide dismutase activity throughout experimental conditions. The activity of apoptosis executioner caspase-3 was significantly increased in the SeT of WT rats treated with 250 μM of TBHP or 10 μM of Cd, an effect not seen in Tg-RGN animals. These results showed that the SeT of Tg-RGN animals displayed lower levels of oxidative stress and increased antioxidant defenses, exhibiting protection against oxidative damage and apoptosis. Moreover, the present findings support the antioxidant role of RGN in spermatogenesis, which may be an important issue of further research in the context of male infertility. Copyright © 2016 John Wiley & Sons, Ltd. Keywords: cadmium; glutathione-S-transferase; oxidative stress; regucalcin; reproductive toxicity; seminiferous tubules; spermatogenesis; superoxide dismutase; TBARS; tert-butyl hydroperoxide Introduction Regucalcin (RGN) is a calcium (Ca 2+ )-binding protein that contrib- utes to Ca 2+ homeostasis by the regulation of plasma membrane, mitochondria and endoplasmic reticulum Ca 2+ -ATPases activity (Yamaguchi, 2000). Besides the function in the modulation of intra- cellular Ca 2+ , the RGN protein has been linked to the control of cell survival and apoptosis, and its antioxidant properties also have been described (Son et al., 2008; Handa et al., 2009; Yamaguchi, 2013). The reactive oxygen species (ROS) may be generated in response to exogenous agents or via endogenous processes and have important physiological roles (Thannickal and Fanburg, 2000). However, when ROS production greatly exceeds the capac- ity of the endogenous cellular antioxidant defense system, oxida- tive stress occurs. The antioxidant role of RGN was effectively demonstrated by studies in the RGN-knockout mice, which display higher levels of oxidative stress than their wild-type (WT) counter- parts (Son et al., 2006). Accordingly, the overexpression of RGN in liver and kidney cells protected against oxidative stress by reduc- ing the production of ROS and increasing the activity of antioxi- dant defense enzymes (Fukaya and Yamaguchi, 2004; Ichikawa and Yamaguchi, 2004; Handa et al., 2009). Oxidative stress is one of the main harmful factors for male re- productive function, and a major cause of male infertility because of its damaging effects on the developing germ cells and sperm function (Aitken and Curry, 2011). Therefore, successful spermato- genesis and the achievement of optimal sperm function require protection from increased levels of oxidative stress. The mecha- nisms protecting against oxidative stress depend on the activity of the antioxidant enzyme system, which includes, among others, glutathione-S-transferase (GST) and superoxide dismutase (SOD). It is also known that RGN can modulate the activity of GST and SOD (Fukaya and Yamaguchi, 2004; Handa et al., 2009), and the benefi- cial role of RGN for male germ cells was also indicated, with RGN overexpression protecting germ cells from apoptosis induced by damaging factors (Correia et al., 2014). Moreover, previous findings of our research group showed a higher antioxidant potential and diminished lipid peroxidation in the rat epididymis with increased expression of RGN (Correia et al., 2013). Nevertheless, the antioxi- dant role of RGN in the testis remains to be clarified, which is of the uttermost importance considering germ cells specificities and its high sensitivity to damaging factors. In the present study, we investigated the role of RGN in modu- lating the levels of oxidative stress in the testis by comparing the antioxidant status and the activity of antioxidant enzymes be- tween seminiferous tubules (SeT) of transgenic rats overexpressing RGN (Tg-RGN) and their WT counterparts, after ex vivo culture in the presence or absence of oxidant inducers, tert-butyl hydroper- oxide (TBHP) and cadmium chloride (Cd). The oxidative damage associated with apoptotic cell death in the SeT of Tg-RGN and *Correspondence to: Sílvia Socorro, CICS-UBI, Centro de Investigação em Ciências da Saúde, Faculdade de Ciências da Saúde, Universidade da Beira Interior, Av. Infante D. Henrique, 6200-506 Covilhã, Portugal. E-mail: ssocorro@fcsaude.ubi.pt CICS-UBI - Health Sciences Research Centre, University of Beira Interior, Covilhã, Portugal J. Appl. Toxicol. 2016 Copyright © 2016 John Wiley & Sons, Ltd. Research article Received: 16 January 2016, Revised: 8 March 2016, Accepted: 15 March 2016 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jat.3333