Developmental Expression and Functions of PLCf Figure 6. Indirect immunofluorescence analysis of PLCf during in vitro fertilization in swine. Note the PLCf immunoreactivity (red) in the acrosome region during sperm-zona binding. The immunoreactivity disappears along with acrosome shroud (arrows) when the spermatozoa undergo the acrosome reaction. No PLCf immunoreactivity was detectable on the sperm heads during sperm-oolemma binding and incorporation into the oocyte. The results shown here are from anti-pPLCf while a similar result was observed with anti-EF. DAPI (blue) was used for DNA staining; DIC, Differential Interference Contrast. Bars = 25 mm. doi:10.1371/journal.pone.0033496.g006 oocyte activating factor (SOAF) during spermiogenesis and its appropriate assembly as part of the PAS-PT [9,10]. Failure of oocyte activation is considered to be responsible for 2–3% of failed fertilization cases when ICSI is performed [35]. Therefore, it is crucial to identify SOAF and its potential contribution to infertility, as well as to harness it for infertility treatments. Among the several SOAF candidates, PLCf has been discussed most extensively in the last decade because its cRNA or recombinant protein induces calcium oscillations and oocyte activation when injected into the oocyte [19,36]. Although there is much information about the characteristics of PLCf such as its active domains [37] or expression in different species [18], little is known about its developmental expression during spermiogenesis, and localization in the spermatozoa before and during fertilization process. PLCf’s location is not consistent in spermatozoa between species The importance of SOAF localization to the PT was highlighted when Kimura et al. [38] showed that the incubation of the sperm heads with non-ionic detergent, Triton X-100, removed all sperm membranous and acrosomal components, but left the PT and the oocyte-activating ability of the sperm head intact. The mandatory localization of SOAF to the PT was later confirmed by Perry et al. [39] and Sutovsky et al. [40]. In addition, the latter investigators provided convincing evidence that SOAF was regionalized to the PAS-PT, which is the first part of the PT solubilized on sperm entry into the oocyte cytoplasm. Therefore on the premise that SOAF should not be extractable by non-ionic detergents and should reside in the PT, we were perplexed to find that a 74 kDa protein corresponding to the catalytically active PLCf isoform was extractable with non-ionic detergent from human spermatozoa, while a smaller, presumably inactive PLCf isoform remained in the pellet. Grasa et al. [32] found a similar 74 kDa PLCf band along with other smaller size bands in whole human sperm, while only the 74 kDa band was released to the sperm extract by freeze thaw, which is in agreement with our results. In the mouse, on the other hand, the presumably full size active PLCf isoform was detergent resistant. It was not retained in the sperm head as expected but rather in the sperm tail. Young et al. PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e33496