highlighted by the fact that the Plcf (2/2) knockout mice are not appropriate models to study because of the arrest of spermiogen- esis for unknown reasons [51]. As shown in the case of other SOAF candidate, PAWP, competitive peptides and antibodies were able to block sperm-induced intracellular calcium release and meiotic resumption in amphibians and mammalian oocyte activation, respectively, providing proof that PAWP has a non- redundant role in oocyte activation [14,15]. Identification of SOAF could improve the understanding of the etiology of male infertility and failed fertilization after ART. SOAF could be used as a biomarker for accurate diagnosis of male infertility in couples with unexplained, idiopathic infertility, which comprises up to 20% of all infertility cases [54,55,56]. Some patients with globozoospermia have demonstrated defective or absent PLCf in sperm extracts [22,24]. However, as many other acrosomal and PT proteins are absent in these patients [57], there is no conclusive evidence showing that PLCf is the actual cause of failed fertilization and infertility in these patients. The other important aspect of SOAF is highlighted in couples with low fertilization rate following ICSI, due to sperm failure to activate the oocyte. The availability of an authentic sperm borne oocyte- activating factor in recombinant form could enhance the efficiency of these treatments, at the same time being more efficient and safer than artificial oocyte activation by chemical methods or mRNA/ cRNA injection [58]. The proper developmental analysis is required to make the scientific decision of whether a candidate protein is a SOAF. We strongly recommend the precise SOAF characterization as a prerequisite to determining SOAF candidates and finding the actual role of PLCf in reproduction. In conclusion, the present study shows that PLCf is an intra- acrosomal protein that incorporates into the forming acrosome during spermatid differentiation, but contrary to expectations for a SOAF candidate, is not incorporated into the assembling PAS-PT. We demonstrate, for the first time, the expression of PLCf in mouse epididymis that may result in the acquisition onto sperm head surface. During IVF-induced acrosome reaction on the zona pellucida surface, PLCf disappears and does not appear to be incorporated into the oocyte cytoplasm during fertilization. We propose the possibility of acrosomal, surface and mitochondrial PLCf compartmentalization in the spermatozoa. Our results along with the recent findings from other researchers make it unlikely that this member of phospholipase family is a genuine sperm- borne oocyte activating factor. Materials and Methods All animal works in this study have been conducted according to national guidelines, approved by Queen’s University Anima Care Committee (approval# Oko-2007-007-R3). Ethical approval for research on the human samples in this study was obtained from Queen’s University Health Sciences Research Ethics Board. Written informed consents were obtained from all participants. Antibodies To scrutinize its subcellular localization in spermatozoa, antibod- ies were raised against different regions of PLCf, including novel target epitopes as well as epitopes targeted by antibodies in studies by other researchers. We raised affinity purified polyclonal rabbit anti- serum against the synthetic sequence comprising the EF hands as crucial domains for the function of PLCf [59] in human, mouse and pig (C-KDNDRLKQGRITIEEF-coNH2), named as anti-EF in the text. The second affinity purified polyclonal rabbit anti-serum was raised following the procedures described by Young et al. [30] against two synthetic peptides of hamster, human and mouse PLCf (C- Developmental Expression and Functions of PLCf MEMRWFLSKIQDEFRGGKI-coNH2 and C-CMNKGYRRV PLFSK-coNH2), named as anti-hmPLCf. Additionally for human spermatozoa, we used the polyclonal rabbit antiserum against two peptides of human PLCf (C-RESKSYFNPSNIKE-coNH2; C- ETHERKGSDKRGDN-coNH2), initially obtained from Dr. John Parrington, University of Oxford, and later purchased from Covalab (Villeurbanne, France), named as anti-hPLCf. The immunoblotting and immunocytochemistry experiments were performed using different corresponding antibodies to achieve the most consistent results. In addition to anti-EF for porcine in vitro fertilization, we used polyclonal antiserum against 19-mer peptides of porcine PLCf (C- MENKWFLSMVRDDFKGGK-coNH2), named as anti-pPLCf, raised in rabbits following the procedures described by Kurokawa et al. [41]. Sperm preparation and protein extraction In order to localize the protein in the sperm compartments as accurately as possible, we chose to study different soluble and insoluble fractions of mouse and human sperm extracts. Sperm were obtained from the cauda epididymis of mature CD1 male mice (Charles River, St-Constant, QC, Canada). To extract the membranous components such as plasmalemma, acrosome and inner acrosomal membrane, the washed spermatozoa were treated with non-ionic detergent, 1% NP40 in the presence of 0.01 M phenyl methylsulphonyl fluoride (PMSF). To detach the sperm heads from the tails, the NP40 pellet was washed several times and sonicated with a Vibrocell Sonicator (50 Watt model, Sonics and Materials Inc., Danbury, CT) according to the previous protocol [60]. The efficiency of separation was monitored by phase contrast microscopy until .99% of head-tail junctions were broken. The head/tail mixture was further resuspended in buffer containing 80% sucrose. Following the previously described protocol [60,61], sperm heads and tails were isolated on a sucrose gradient with over 99% purity confirmed by phase microscopy. Furthermore, isolated sperm tails were treated with Triton- dithiothretiol (DTT) mixture (2% Triton X-100, 5 mM DTT, 50 mM Tris-HCl, pH 9) in order to denude the mid-piece of the mitochondrial sheath [62]. Human spermatozoa were donated by three normal fertile males. The semen samples were allowed to liquefy, then washed and pooled together. The NP40 extraction was performed as described above. To mitigate the effects of repeated freeze-thaw process, all sperm samples were treated fresh and sperm fractions were used immediately kept in 280uC until further use. Immunoblotting Sperm samples were dissolved in reducing sample buffer containing 2% SDS and 5% b-mercaptoethanol. Equal amounts of sperm extracts (16107 sperm/lane) were loaded and resolved on 10 or 12% SDS-PAGE. Proteins were transferred to either nitrocellulose (Schleicher and Schuell, Dassel, Germany) or polyvinylidene fluoride (PVDF; Millipore, Mississauga, ON) membranes. The immunoreactivity on western blots was detected with peroxidase labeled goat anti-rabbit IgG (Vector Laboratories, Inc., Burlingame, CA) diluted 1:25,000 (v/v) and further use of enhanced chemiluminescent substrate (Pierce, Rockford, IL) with exposure to X-ray films. Immunoproxidase staining for light microscopy The paraffin-embedded human testicular tissue, obtained from cancer patients who underwent total orchiectomy in Kingston General Hospital, Queen’s University, along with mouse testicular and epididymal tissues were processed for immunoperoxidase staining according to previously described protocol [63]. Following deparaffinization in xylene and hydration through a concentration PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e33496