Animal Reproduction Science 145 (2014) 29–39 Contents lists available at ScienceDirect Animal Reproduction Science jou rn al hom epage : w ww.elsevier.com/locate/anir eprosci Thiol-disulfide proteins of stallion epididymal spermatozoa G.M. Dias a , M.L. López a,∗ , A.T.S. Ferreira b , D.A. Chapeaurouge b , A. Rodrigues a , J. Perales b , C.A. Retamal a a Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, UENF, Campos dos Goytacazes, RJ, Brazil b Laboratório de Toxinologia, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil a r t i c l e i n f o Article history: Received 20 September 2013 Received in revised form 14 December 2013 Accepted 17 December 2013 Available online 31 December 2013 Keywords: Equus caballus spermatozoa Sperm maturation Sulphydrylated proteins Thiol oxidation a b s t r a c t Thiol groups of cysteine residues represent redox centers involved in multiple biological functions. It has been postulated that changes in the redox status of mammalian epididy- mal spermatozoa contribute to the sperm maturation process. The present work shows the thiol-disulfide protein profile of stallion epididymal spermatozoa achieved by two- dimension electrophoresis and MALDI-TOF/TOF mass spectrometry of proteins labeled with a thiol-reactive fluorescent tag, monobromobimane. Our results have shown the formation of disulfide bonds in several sperm protein fractions during the epididymal maturation process. The majority of the oxidized thiol sperm proteins identified correspond to struc- tural molecules of the flagellum (as the outer dense fiber-1 protein – ODF1), followed by glycolytic enzymes (as glyceraldehyde-3-phosphate dehydrogenase spermatogenic), antioxidant protectors (as glutathione S-transferase and phospholipid hydroperoxide glu- tathione peroxidase – PHGPx). The magnitude of the thiol oxidation differs between proteins, and was more drastic in polypeptides with molecular weights of up to 33 kDa, identified as ODF1 and PHGPx. A kinase anchor protein, a voltage-dependent anion channel protein and a zona pellucida-binding protein were also found in the polypeptide samples that contained oxidized SH groups. These proteins may be modified or controlled by the mechanisms involved in the cysteine-redox changes, corroborating the belief that a correct degree of protein oxidation is required for the stabilization of sperm structure, protection against oxidative damage, induction of progressive sperm motility and fertilization. Published by Elsevier B.V. 1. Introduction The thiol groups of cysteine residues represent redox centers involved in protein structure, enzyme catalysis, signal transduction and regulation of the transcriptional activity of different cell types (Barford, 2004; Fomenko et al., 2008; Jones, 2010). The redox-based regulation of gene expression has emerged as a fundamental regulatory mechanism in cell biology. A variety of proteins, includ- ing transcription factors, molecular chaperones, protein ∗ Corresponding author. Tel.: +55 22 27234705. E-mail addresses: mlopez@uenf.br, mlopezuenf@gmail.com (M.L. López). tyrosine phosphatases and protein tyrosine kinases, are activated or regulated via redox processes. Conformational changes and/or other post-translational modifications may result from the oxidation of thiols within the protein itself or belonging to interacting proteins, thus mediating diverse responses according to the cell physiological state (Jones, 2010; Monteiro et al., 2008; Sen, 2000). During the epididymal transit, spermatozoa complete a maturation process that enables these cells to inter- act with the oocyte at the appropriate time and place. This process is the result of a complex cascade of post- translational changes that involve the rearrangement of the sperm membrane components, the induction of chromatin condensation, the stabilization of the tail components, the processing of different proteins and others modifications 0378-4320/$ – see front matter. Published by Elsevier B.V. http://dx.doi.org/10.1016/j.anireprosci.2013.12.007