l-Arginine promotes capacitation and acrosome reaction in cryopreserved bovine spermatozoa Cristian O’Flaherty a, * , Pablo Rodriguez a , Sudha Srivastava b a Biochemistry Area, School of Veterinary Sciences, University of Buenos Aires, Av. Chorroarı ´n 280 (C1427CWO) Buenos Aires, Argentina b National Facility for High Field NMR, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai-400005, India Received 20 October 2003; received in revised form 22 June 2004; accepted 24 June 2004 Available online 23 July 2004 Abstract Sperm capacitation and acrosome reaction are essential for fertilization and they are considered as part of an oxidative process involving superoxide and hydrogen peroxide. In human spermatozoa, the amino acid l-arginine is a substrate for the nitric oxide synthase (NOS) producing nitric oxide (NO!), a reactive molecule that participates in capacitation as well as in acrosome reaction. l-Arginine plays an important role in the physiology of spermatozoa and has been shown to enhance their metabolism and maintain their motility. Moreover, l- arginine has a protective effect on spermatozoa against the sperm plasma membrane lipid peroxidation. In this paper, we have presented, for the first time, the effect of l-arginine on cryopreserved bovine sperm capacitation and acrosome reaction and the possible participation of NOS in both processes. Frozen-thawed bovine spermatozoa have been incubated in TALP medium with different concentrations of l- arginine and the percentages of capacitated and acrosome reacted spermatozoa have been determined. l-Arginine induced both capacitation and acrosome reaction. NO! produced by l-arginine has been inhibited or inactivated using NOS inhibitors or NO! scavengers in the incubation medium, respectively. Thus, the effect of NOS inhibitors and NO! scavengers in capacitated and non-capacitated spermatozoa treated with l-arginine has also been monitored. The data presented suggest the participation of NO!, produced by a sperm NOS, in cryopreseved bovine sperm capacitation and acrosome reaction. D 2004 Elsevier B.V. All rights reserved. Keywords: l-Arginine; Nitric oxide; Nitric oxide synthase; Sperm capacitation; Acrosome reaction 1. Introduction Mammalian spermatozoa must undergo a series of membranous and metabolic changes before they can fertilize the egg. These physiological changes represent a complex process called capacitation [1–3]. Various in vitro studies have indicated that the process of capacitation is biochemical in nature. Capacitation regulates transient changes in the sperm motility pattern, termed hyper- activation, and enables the exocytotic event of acrosome reaction, an essential event for oocyte fertilization [4]. Though the capacitation is achieved synergistically and efficiently in the female reproductive tract, it can also be accomplished in vitro in various well-defined media for several species of mammals. Nitric oxide (NO!), a highly reactive oxygen species (ROS), has been found in several physiological systems and plays a decisive role in regulating multiple functions within the male as well as female reproductive systems. It is derived from l-arginine by the enzyme nitric oxide synthase (NOS) [5], using oxygen and different cofactors such as nicotinamide adenine dinucleotide phosphate, flavin mononucleotide, flavin adenine dinucleotide, and tetrahydrobiopterin, as well as calmodulin and calcium [6]. These enzymes are found on the acrosome and tail region of non-capacitated spermatozoa [7,8]. At low concentra- 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.06.020 * Corresponding author. Present address: Urology Research Labora- tory, Room H6.44, Royal Victoria Hospital, McGill University, 687 Ave des Pins Ouest, Montreal, Quebec, Canada H3A 1A1. Tel.: +1 514 842 1231x35429. E-mail address: coflaher@yahoo.com (C. O’Flaherty). Biochimica et Biophysica Acta 1674 (2004) 215 – 221 www.bba-direct.com