PROOF ONLY CSIRO PUBLISHING www.publish.csiro.au/journals/rfd Reproduction, Fertility and Development, 2008, 20, 1–10 Mitochondrial activity and forward scatter vary in necrotic, apoptotic and membrane-intact spermatozoan subpopulations F. Martínez-Pastor A,B,C , M. R. Fernández-Santos A,B , E. del Olmo A,B , A. E. Domínguez-Rebolledo A,B , M. C. Esteso A,B , V. Montoro and J. J. Garde A,B A Biology of Reproduction Group, National Wildlife Research Institute, Higher Council for Scientific Research, University of Castilla-La Mancha, Regional Government of Castilla-La Mancha, 0B. 0G. A., Albacete 02071, Spain. B Institute for Regional Development, University of Castilla-La Mancha, 0B. 0G. A., Albacete 02071, Spain. C Corresponding author. Email: felipe.martinez@uclm.es Abstract. In the present study, we have related mitochondrial membrane potential ( m ) and forward scatter (FSC) to apoptotic-related changes in spermatozoa. Thawed red deer spermatozoa were incubated in synthetic oviductal fluid medium (37 ◦ C, 5% CO 2 ), with or without antioxidant (100 μm trolox). At 0, 3, 6 and 9 h, aliquots were assessed for motility and were stained with a combination of Hoechst 33342, propidium ioide (PI),YO-PRO-1 and Mitotracker Deep Red for flow cytometry. The proportion of spermatozoa YO-PRO-1+ and PI+ (indicating a damaged plasmalemma; DEAD) increased, whereas that of YO-PRO-1− and PI− (INTACT) spermatozoa decreased. The proportion of YO- PRO-1+ and PI− spermatozoa (altered plasmalemma; APOPTOTIC) did not change. Both DEAD and APOPTOTIC spermatozoa had low  m . Most high- m spermatozoa were INTACT, and their proportion decreased with time.The FSC signal also differed between different groups of spermatozoa, in the order APOPTOTIC > DEAD > INTACT/low  m > INTACT/high  m ; however, the actual meaning of this difference is not clear. APOPTOTIC spermatozoa seemed motile at 0h, but lost motility with time. Trolox only slightly improved the percentage of INTACT spermatozoa (P< 0.05). The population of APOPTOTIC spermatozoa in the present study may be dying cells, possibly with activated cell death pathways (loss of  m ). We propose that the sequence of spermatozoon death here would be: (1) loss of  m ; (2) membrane changes (YO-PRO-1+ and PI−); and (3) membrane damage (PI+). INTACT spermatozoa with low  m or altered FSC may be compromised cells. The present study is the first that directly relates membrane integrity, apoptotic markers and mitochondrial status in spermatozoa. The results of the present study may help us understand the mechanisms leading to loss of spermatozoon viability after thawing. Additional keywords: cell volume, flow cytometry, membrane changes, mitochondrial membrane potential, sperm death, YO-PRO-1. Introduction Even though spermatozoa may lack a complete apoptotic mech- anism (Weil et al. 1998), many studies have confirmed that at least part of the apoptotic pathway could be active, or poten- tially active, in spermatozoa (Paasch et al. 2004a; Grunewald et al. 2008; Marti et al. 2008). In fact, active caspases, pro- teases functioning as apoptotic mediators, have been detected in spermatozoa under various conditions (Paasch et al. 2003, 2004a, 2004b). Previous studies have described the effects of apoptosis in spermatozoa in terms of membrane changes, loss of mitochondrial membrane potential ( m ) and vol- ume deregulation. Changes in membrane permeability have been described as a typical apoptotic event in many cell types (Idziorek et al. 1995). Although spermatozoa can undergo phys- iological changes on the plasmalemma, due to capacitation, it was soon discovered that some of these changes were linked to apoptotic markers (Waterhouse et al. 2004; Martin et al. 2005). Spermatozoa affected by these apoptotic-like events exhibit membrane changes that apparently herald cell death (Peña et al. 2005). Loss of  m associated with apoptosis has been found in spermatozoa (Martin et al. 2005; Aziz et al. 2007; Grunewald et al. 2008). The presence of active mitochondria is important because they participate in many regulatory and maintance pro- cesses (Aitken et al. 2007). Therefore, we can consider that apoptotic spermatozoa may eventually lose viability because © CSIRO 2008 10.1071/RD08002 1031-3613/08/050001 AUTHORS’ PAGE PROOFS: NOT FOR CIRCULATION