MOLECULAR REPRODUCTION AND DEVELOPMENT 73:1179–1194 (2006) Hexose-Specificity of Hexokinase and ADP-Dependence of Pyruvate Kinase Play Important Roles in the Control of Monosaccharide Utilization in Freshly Diluted Boar Spermatozoa ANTONIO MEDRANO, 1 NU ´ RIA GARCI ´ A-GIL, 2 LAURA RAMIO ´ , 1 MA. MONTSERRAT RIVERA, 1 JOSEP MA. FERNA ´ NDEZ-NOVELL, 3 ALFREDO RAMI ´ REZ, 4 ALEJANDRO PEN ˜ A, 1 MA. DOLORS BRIZ, 2 ELISABET PINART, 2 ILONA I. CONCHA, 4 SERGI BONET, 2 TERESA RIGAU, 1 AND JOAN E. RODRI ´ GUEZ-GIL 1 * 1 Department Animal Medicine and Surgery, Unit of Animal Reproduction, School of Veterinary Medicine, Autonomous University of Barcelona, Bellaterra, Spain 2 Biotechnology of Porcine Reproduction Unit, University of Girona, Campus de Montilivi, Girona, Spain 3 Department of Biochemistry and Molecular Biology, University of Barcelona, and Institut de Recerca Biome´dica, Barcelona, Spain 4 Institute of Biochemistry, Faculty of Sciences, Campus Isla Teja, Universidad Austral de Chile; Valdı´via, Chile ABSTRACT Incubation of boar sperm from fresh ejaculates in a minimal medium with 10 mM glucose induced a fast and intense activation of glycolysis, as indicated by the observed increases in the intracellular levels of glucose 6-phosphate (G 6-P) and ATP and the rate of formation of extracellular L-lactate. The effect of glucose was much more intense than that induced by fructose, sorbitol, and mannose. The greater utilization of glucose was related to a much greater sensitivity to hexokinase when compared with the other monosaccharides. Thus, the presence of 0.5 mM glucose induced total hexokinase activity in supernatants from sperm extracts of 1.7 Æ 0.1 mIU/mg protein, while the same concentration of both fructose, mannose, and sorbitol induced total hexokinase activity from 0.3 Æ 0.1 mIU/mg protein to 0.60 Æ 1 mIU/mg protein. Kinetic analysis of the total pyruvate kinase activity indicated that this activity was greatly depen- dent on the presence of ADP and also showed a great affinity for PEP, with an estimated Km in supernatants of 0.15–0.20 mM. Immunological location of proteins closely related to glycolysis, like GLUT-3 hexose trans- porter and hexokinase-I, indicated that these proteins showed the trend to be distributed around or in the cellular membranes of both head and midpiece in a grouped manner. We conclude that glycolysis is regulated by both the specific availability of a concrete sugar and the internal equilibrium between ATP and ADP levels. Furthermore, localization of proteins in- volved in the control of monosaccharide uptake and phosphorylation suggests that glycolysis starts at con- crete points in the boar-sperm surface. Mol. Reprod. Dev. 73: 1179–1194, 2006. ß 2006 Wiley-Liss, Inc. Key Words: boar spermatozoa; hexoses; hexoki- nase; pyruvate kinase; energy metabolism INTRODUCTION In the last several years an increasing amount of information about the mechanism/s by which boar sperm manages its energy levels has been published. This information is of paramount importance in order to understand the mechanisms underlying the boar sperm’s ability to survive and to adapt to the external conditions which often are very aggressive to sperm survival. Moreover, boar sperm has to adapt to sudden changes in these external conditions, associated, for instance, with their rapid passing through the female genital tract, from cervix to oviduct. From a practical point of view, information about mechanisms of energy management is also very important to optimize boar- sperm conservation, both in refrigeration and in freez- ing. This could be of the greatest impact in further improving the worldwide application of artificial inse- mination (AI) techniques in pig production. All of this explains the importance underlying the study of this specific aspect of boar-sperm function. Boar sperm from fresh ejaculates is able to utilize a wide variety of substrates to obtain energy. Some of these are nonmonosaccharides, such as lactate (Jones, 1997), pyruvate (Jones, 1997), glycerol, and glycerol 3-phosphate (Jones et al., 1992). However, monosac- charides seem to be the main energy source and, in this ß 2006 WILEY-LISS, INC. Grant sponsor: Ministry of Science and Technology, Spain; Grant number: AGL2001-2568. *Correspondence to: Joan E. Rodrı ´guez-Gil, Department Animal Medicine and Surgery, Unit of Animal Reproduction, School of Veterinary Medicine. Autonomous University of Barcelona, E-08193 Bellaterra, Spain. E-mail: juanenrique.rodriguez@uab.es Received 21 September 2005; Accepted 10 January 2006 Published online 27 June 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mrd.20480