[30] showed a similar pattern of PLCf immunoblotting in mouse where a 58 kDa band was extracted by Triton X-100, while the remaining PLCf-reactive bands including the presumed active PLCf band remained in the pellet [30]. However, they didn’t further analyze whether the 74 kDa band belonged to the sperm head or tail. Similar to our findings in mouse, Bedford-Guaus et al. [28] immunolabelled the catalytically active PLCf isoform in equine sperm tails. The sperm heads and tails were separated by sonication and centrifugation in this study, while no detergent extraction was performed. Overall, our findings show that the active, full size ,74 kDa PLCf isoform is localized to sperm fractions other than PT. We detected smaller size PLCf-reactive bands (50–60 kDa) which have been observed in other studies and often described as breakdown products, unrelated cross-reactive proteins [22,28,30,32] or splicing variants [36]. Kurokawa et al. performed FPLC fractionation of porcine sperm extracts and, remarkably, detected immunoreactive full size PLCf in a Ca2+ inactive fraction, while several fractions capable of inducing oscillations were devoid of functional PLCf [41]. An alternative explanation for the bands below 74 kDa is that they represent other PLCf isoforms. An isoform of PLCf (NYD-SP27) with a size of approximately 55 kDa has been described recently which is localized to the sperm head as well as human pancreas [42,43,44]. This isoform lacks the functionally important EF domains and is suggested to act as an inhibitor of PLC family kinases. NYD-SP27 is proposed to promote capacitation when it detaches from sperm head [44]. We recommend further characterization of the undersized PLCf-reactive bands as they may shed light on roles of sperm PLCf irrelevant to oocyte activation. PLCf is secreted as a component of the acrosome during spermiogenesis Developmentally, the assembly of sperm PAS-PT occurs in elongating spermatids by microtubular manchette transport, and is independent of acrosomal formation [45,46,47]. Therefore, to be considered part of the PAS-PT, a SOAF candidate should be shown to originate in elongating spermatids during spermiogen- esis. Contrary to this expectation, our immunocytochemical study shows, for the first time, that human and mouse PLCf is secreted by the Golgi complex within proacrosomic granules that later fuse to form the AV. Interestingly, PLCf in human and mouse appears to diminish from sperm acrosome towards the end of spermio- genesis. It is unlikely that this diminution in PLCf concentration was due to a hidden epitope, as the inner acrosomal membrane protein IAM38 [48], which was used as a control with an identical origin to PLCf, remained intense during spermiogenesis (data not shown). The expression of PLCf mRNA has been shown in different phases of spermatogenesis among different species [25,26,27,30,49]. To our knowledge, there is only one study, in equine testis that shows PLCf translation in the round spermatids [28] and one in the mouse that is inconclusive as to where the protein originates because only one of 12 stages of the cycle of seminiferous epithelium is represented at very low magnification [50]. Besides our immunolocalization of PLCf in the acrosome, our fractionation/immunoblotting data indicates a sperm mitochon- drial source of PLCf. A recent study (Proceedings of 11th International Symposium on Spermatology) shows that Plcf (2/2) knockout mice demonstrate arrest of spermiogenesis at the level of round spermatids [51], a time period during spermiogenesis, according to our analysis, when PLCf levels are highest. We recommend that consideration be given to PLCf involvement in mitochondrial metabolism and acrosomal biogenesis during spermiogenesis. Developmental Expression and Functions of PLCf Epididymal cells secrete PLCf which associates with the sperm head surface We detected PLCf immunofluorescence mainly in the post- acrosomal region of mouse spermatozoa. In human spermatozoa, we localized it to both acrosomal and postacrosomal regions. The localization and intensity of labeling was similar in both permeabilized and non-permeabilized spermatozoa, suggesting the binding of PLCf to the sperm surface. In addition, non-ionic detergent extraction coincided with the disappearance of im- munolabelling, establishing that PLCf was not a constituent of the detergent resistant perinuclear theca fraction which harbors SOAF. In mouse, PLCf was localized by immunofluorescence to the PAS-PT and acrosomal regions of cauda-epididymal sperma- tozoa [29,30]. Using an identical antibody as Young et al. [30], we could not replicate their results, where PLCf was detected in PAS- PT after mouse sperm extraction with non-ionic detergent Triton X-100. In human, PLCf has been localized over acrosomal, equatorial and postacrosomal regions of the ejaculated sperm head [32]. In equine spermatozoa, it was localized to the acrosome, equatorial region, and in the connecting piece and the principal piece of the sperm tail [28]. Because the studies above did not compare the immunolocalization between permeabilized and non- permeabilized sperm they do not distinguish whether PLCf is within these components or on the membrane surface covering them. The PLCf immunofluorescence findings prompted us to hypothesize the epididymal expression of this sperm protein. We performed RT-PCR, in situ hybridization and immunocytochem- istry to analyze PLCf expression at the mRNA and protein levels, respectively. We demonstrated high level of mRNA expression and protein secretion in the mouse epididymis, both in adult and prepubertal males. Recent studies show PLCf expression in mouse brain [52] as well as the puffer fish ovary and brain [53]. To our knowledge, this is the first time that PLCf protein is reported in the mouse epididymis. These findings negate the thesis of testis specific origin of PLCf. We propose that the signal detected on the head region of mature spermatozoa is most probably from the epididymal sources of PLCf. This is an important consideration because the sperm acrosome and surface proteins are removed early during the fertilization, i.e. prior to sperm-oolemma fusion and sperm incorporation into the ooplasm, at which time the intact sperm PT becomes the only possible source of SOAF for inducing calcium oscillation and oocyte activation [9,38,40]. Spermatozoon lacks PLCf when it fuses with oolemma and enters ooplasm Using established porcine IVF model, we demonstrated the pattern of PLCf localization during in vitro fertilization for the first time and showed that PLCf is mainly observed on the porcine sperm acrosomal region before fertilization. After the acrosome reaction, it is depleted and released together with acrosomal shroud. We were unable to detect any PLCf in the postacrosomal sheath at an early stage of sperm incorporation, when its contents were dispersed into the ooplasm and activated oocyte. This is in stark contrast with well documented, easily traceable release of PAS-PT-resident signalling protein PAWP in the ooplasm during porcine fertilization [14]. Fertilization biology will benefit from further research aimed to understand PLCf’s role in the acrosome reaction during fertilization. Another critical step towards scrutinizing the currently proposed role of PLCf in oocyte activation is to block the ‘sperm’ induced calcium oscillations during in vitro fertilization and after ICSI by PLCf relevant antibodies or competitive peptides. The importance of this step is PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e33496