Nuclear status of immature and mature stallion spermatozoa G.M. Dias, C.A. Retamal, L. Tobella, A.C.V. Arnholdt, M.L. Lo ´pez * Centro de Biocie ˆncias e Biotecnologia, LBCT, Setor Biologia da Reproduc ¸a ˜o, Universidade Estadual do Norte Fluminense, Av. Alberto Lame ˆgo 2000, Horto, Campos dos Goytacazes CEP: 28013600, RJ, Brazil Received 25 August 2005; accepted 16 October 2005 Abstract ’The highly packed chromatin of mature spermatozoa results from replacement of somatic-like histones by highly basic arginine- and cysteine-rich protamines during spermatogenesis, with additional conformational changes in chromatin structure during epididymal transit. The objective of the present study was to compare the nuclear characteristics of immature and mature epididymal stallion spermatozoa, using a variety of experimental approaches. Resistance to in vitro decondensation of chromatin, following exposure to SDS-DTT and alkaline thioglycolate, increased significantly in mature spermatozoa. Evaluation of the thiol- disulfide status (monobromobimane labeling) demonstrated that immature cells obtained from ductulli efferentes contained mostly thiol groups, whereas these groups were oxidized in mature cells collected from the cauda epididymidis. Based on atomic absorption spectrophotometry, maturation of stallion spermatozoa was accompanied by a 60% reduction in the Zn 2+ content of sperm cells, concomitant with increased concentrations of this ion in epididymal fluid. Furthermore, the degree of disulfide bonding was inversely correlated with susceptibility of chromatin to acid denaturation (SCSA). Collectively, these data were consistent with the hypothesis that maturation of stallion spermatozoa involves oxidation of sulphydryl groups to form intra- and intermolecular disulfide links between adjacent protamines, with loss of zinc as an integral feature. These changes endow mechanical and chemical resistance to the nucleus, ensuring efficient transmission of the paternal genome at fertilization. # 2005 Elsevier Inc. All rights reserved. Keywords: Sperm maturation; Chromatin decondensation; Zinc 2+ ; Thiol-disulfide status; Stallion 1. Introduction During spermiogenesis, the nucleus of spermatids undergoes complex morphological, biochemical and physiological alterations. Their shape, size and con- densation state change dramatically, due to exchange of histones by transition proteins and highly basic arginine- and cysteine-rich protamines [1,2]. These changes in the complex DNA-proteins eliminate the nucleosomal organization of chromatin, resulting in a tightly packed toroidal-like structure, containing up to 60 kb of DNA, in which the transcription and repair activities are inactivated [3,4]. The primary factor that induces compaction is thought to be protamine binding; it neutralizes the negative charge on the phosphodiester backbone of DNA [5]. In vitro studies of the kinetics of DNA condensation and decondensation induced by protamines and synthetic peptides have shown that the number of clustered arginine residues present in the DNA binding domain is the most important factor affecting the condensation and stability of the DNA-protamine complex, prior to the formation of inter-protamine disulfide cross-links [4]. Although the exact structure of the complex has not been determined, spectroscopic studies suggested that protamine binds to DNA in an extended conformation, with a footprint that covers 11 bp [5]. Protamine binding experiments, conducted in the presence of Zn 2+ , suggested that a zinc finger would www.journals.elsevierhealth.com/periodicals/the Theriogenology 66 (2006) 354–365 * Corresponding author. Tel.: +55 22 27261690. E-mail address: mlopez@uenf.br (M.L. Lo ´pez). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.10.024