Ram seminal plasma proteome and its impact on liquid preservation of spermatozoa C. Soleilhavoup a, b, c , d , G. Tsikis a, b, c , d , V. Labas a, b, c , d, e , G. Harichaux a, b, c , d, e , P.L. Kohnke a, b, c , d , J.L. Dacheux a, b, c , d , Y. Guérin a, b, c , d , J.L. Gatti a, b, c , d , S.P. de Graaf f , X. Druart a, b, c , d, ⁎ a INRA, UMR 85 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France b CNRS, UMR7247, F-37380 Nouzilly, France c Université François Rabelais de Tours, F-37000 Tours, France d IFCE, Institut Français du Cheval et de l'Equitation, F-37380 Nouzilly, France e INRA, Plate-forme d'Analyse Intégrative des Biomolécules (PAIB), Laboratoire de Spectrométrie de Masse, F-37380 Nouzilly, France f Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia ARTICLE INFO ABSTRACT Article history: Received 7 May 2014 Accepted 7 July 2014 Seminal plasma is composed of secretions from the epididymis and the accessory sex glands and plays a critical role in the fertilising ability of spermatozoa. In rams, analysis of seminal plasma by GeLC–MS/MS has allowed the identification of more than 700 proteins, including a high abundance of Binder of Sperm family proteins (BSP1, BSP5, SPADH1, SPADH2), the spermadhesin family (bodhesin2), lactoferrin and newly identified proteins like UPF0762 (C6orf58 gene). When spermatogenesis was stopped by scrotal insulation, changes in the proteome profile revealed the sperm origin of 40 seminal proteins, such as glycolysis pathway enzymes, the chaperonin containing TCP1 (CCT) complex and the 26S proteasome complex. Sperm mobility after liquid preservation (24 h in milk at 15 °C) is male dependent and can be correlated to differences in the seminal plasma proteome, detected by spectral counting. The negative association of zinc alpha-2 glycoprotein (ZAG) with semen preservation was confirmed by the use of recombinant human ZAG, which induced an increase in mobility of fresh sperm, but then decreased sperm mobility after 24 h of incubation. Several sperm membrane proteins interacting with the cytoskeleton, glycolysis enzymes and sperm-associated proteins involved in capacitation correlated with better liquid storage and can be considered as seminal biomarkers of sperm preservation. Biological significance Extensive analysis of the ram seminal plasma proteome reveals a complex and diverse protein composition. This composition varies between males with different sperm preservation abilities. Several proteins were shown to originate from the spermatozoa and positively correlate with sperm liquid preservation, indicating that these proteins can be traced as sperm Keywords: Seminal plasma Spermatozoa Zinc alpha glycoprotein Preservation Proteome Spectral counting JOURNAL OF PROTEOMICS 109 (2014) 245 – 260 ⁎ Corresponding author at: UMR INRA 85, CNRS 7247-Université François Rabelais-IFCE, Unité de Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique, Centre de Recherches Val de Loire, 37380 Nouzilly, France. Tel.: +33 2 47 42 79 49. E-mail address: xavier.druart@tours.inra.fr (X. Druart). http://dx.doi.org/10.1016/j.jprot.2014.07.007 1874-3919 © 2014 Elsevier B.V. All rights reserved. Available online at www.sciencedirect.com ScienceDirect www.elsevier.com/locate/jprot