while postacrosomal WW binding protein (PAWP) [14,15,16], citrate synthase [17] and phospholipase C zeta (PLCf) [18] are presently under investigation. PLCf is a member of phospholipase family that was suggested as a SOAF candidate in 2002 [19]. It was demonstrated that microinjection of PLCf cRNA or recombinant protein initiates intracellular calcium release and oocyte activation [19,20]. In a proportion of patients with globozoospermia, where multiple defects such as absence of acrosome and PAS-PT, impaired sperm cytoskeleton and sperm inability to activate oocyte are observed [21], PLCf was shown to be absent or defective [22,23]. Sperm from these patients failed to activate the mouse oocyte, unless artificially activated by calcium ionophore [24]. PLCf mRNA is present in a range of spermatogenetic phases (i.e. spermatocytes, round and elongating spermatids) dependent on species [19,25,26,27]. However, there is no information about the developmental expression of PLCf protein during mammalian spermatogenesis, except for equine where it is localized to the round spermatids [28]. In the spermatozoa, it has been localized to varied locations including the post acrosomal, acrosomal, equatorial and mid-piece regions [28,29,30,31,32]. In addition neither PLCf localization during fertilization nor its non- redundancy as a SOAF has been demonstrated yet [33]. Despite past efforts, there is no evidence that PLCf is released from sperm head PT into the oocyte cytoplasm immediately after sperm- oolemma fusion that coincides with oocyte activation. In order to clarify whether PLCf meets these crucial characteristic requirements of SOAF, we investigated the developmental expression of the protein within the testis and epididymis. We identified the pattern of PLCf secretion during spermatogenesis and epididymal maturation, and followed its fate during in vitro fertilization. Based on our results, we suggest that PLCf does not meet some of the important criteria to be considered as SOAF. Results Localization of PLCf in sperm head and tail fractions Immunoblotting of the whole mouse cauda-epididymal sperm extracts with a peptide affinity purified anti-PLCf antibody revealed four major bands of approximately 74, 55, 45 and 43 kDa (Fig. 1A, WS). Non-ionic detergent, NP40 extracted a faint 58 kDa band while the other major bands, including the presumably functional 74 kDa PLCf band remained in the pellet (Fig. 1A). This finding prompted us to separate the NP40 extracted sperm heads and tails by sonication and sucrose gradient centrifugation, in order to locate in which fraction the detergent insoluble PLCf bands resided. Surprisingly, PLCf immunoreac- tivity was not observed in the isolated sperm heads or sonicated supernatant, but rather in the isolated sperm tails (Fig. 1B). Incubation of the isolated tails with Triton X100-DTT completely extracted all PLCf immunoreactive bands, indicating a mito- chondrial sheath origin (Fig. 1B). In the human spermatozoa, two distinct PLCf–reactive bands of approximately 74- and 45- kDa were observed in whole ejaculated sperm extracts (Fig. 1C, WS). However, the 74 kDa band and most likely its breakdown product, a 55 kDa protein band, were extracted by NP40, while the detergent insoluble 45 kDa band remained in the pellet (Fig. 1C). Developmental expression of PLCf during spermiogenesis by light and electron microscopy We performed immunoproxidase staining on mouse and human paraffin embedded testicular tissues. In mouse, PLCf was detected Developmental Expression and Functions of PLCf Figure 1. Immunoblotting of PLCf after differential extraction of mouse and human spermatozoa. (A) Detergent extraction of mouse cauda epididymal spermatozoa. The ,74 kDa band correspond- ing to the functional isoform of PLCf (arrow) was detected in whole sperm (WS) and was resistant to NP40 extraction. Anti-hmPLCf was used to detect the signal with a similar result obtained with anti-EF. (B) The NP40 resistant sperm fraction (i.e., pellet in panel A) was sonicated and sperm heads and tails were separated through a sucrose gradient. The 74 kDa active isoform was found only in the tail fraction (arrow). Further incubation of the tail fraction with Triton x-100/DTT (Tail Tx- DTT) extracted all of the PLCf bands, suggesting the mitochondrial sheath of the mid piece as the origin of PLCf in the sperm tail. Anti-EF was used to detect the signal with a similar result obtained with anti- hmPLCf. Lanes were run on the same gel but were non-contiguous. (C) Human sperm extraction by NP40. The functional isoform (arrow) was detergent extractable as shown in NP40 lane. Anti-hmPLCf was used for detection. A similar result was observed with anti-EF and anti-hPLCf. WS, Whole sperm; Sc.Sn, Sonication supernatant. doi:10.1371/journal.pone.0033496.g001 at the stage III of spermatogenesis in the acrosomic vesicles (AV) of step 3 round spermatids. The immunoreactivity was concentrated in the acrosomic granule, located at the center of AV (Fig. 2A). PLCf immunostaining remained intense over the acrosomal cap in stage VII and resided in the head of elongating spermatids in stages XI and XII. Surprisingly, by step 14 spermatids (stages II and III of spermatogenesis), the intensity of immunostaining associated with elongated spermatid nuclei diminished substan- tially. Little or no PLCf immunostaining was detected in spermatids at the end of spermiogenesis, except in opportune sections through the sperm tails (data not shown). In human testicular tissues, immunostaining localized PLCf to the AVs of step 2 spermatids in stage II of the six stage cycle of the human seminiferous epithelium (Fig. 2B). In step 4 spermatids (stage IV) immunostaining was concentrated over the fully formed acrosome, covering the apical half of the spermatid nucleus. Importantly, little immunoreactivity was found else- where in the epithelium and elongated spermatids (Fig. 2C). In both species, saturation of antibodies with oligopeptides used to raise the immune serum resulted in elimination of immunostain- ing over the round spermatids of seminiferous tubule sections in similar stages of development, confirming the specificity of reactions (Fig. S1). To confirm and clarify immunoperoxidase findings, anti-PLCf immunogold labelling was performed on mouse testicular sections prepared for electron microscopy. PLCf was detected in the proacrosomic granules or vesicles within the Golgi complex of steps 1 and 2 round spermatids (Fig. 3A, B). Through steps 2–4, the PLCf-containing granules fused together to from the AV, which docked onto the spermatid nucleus (Fig. 3C). During the acrosome capping process in steps 5–7, the immunogold labeling intensity shifted from the acrosomic granule to the acrosomal collar, suggesting redistribution of PLCf from the acrosomic granule (Fig. 3D). Throughout the last steps of spermiogenesis there was a noticeable diminution in the intensity of acrosome labelling (Fig. 3E). PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e33496